By Ananda on Wednesday, February 12, 2003 - 12:34 pm:
I was really suprised today when i was trying to prepare a HPLC mobile phase. I was diluting 100 mL of 100mM KH2PO4 (pH 4.4) in 400 mL of ACN in order to prepare a 20 mM phosphate buffer in 80% acetonitrile. At 2 different pHs, I noticed phosphate was precipitating out and at pH 4.4 it was worse than pH 6.5.
Any ideas and suggestions to make a phophate buffer in acetonitrile at these pHs??. I have used K2HPO4 in ACN without any problems before. So if one want to prepare a phophate buffer at low pHs (2, 4.4 and 6) what phophate is the best??? (I personally dont like to use 50% K2HPO4 and KH2PO4 even though it is the best way??)
Your comments and suggestions are mnuch appreciated!!!!!!!!!
By Mike on Wednesday, February 12, 2003 - 12:45 pm:
worse comes to worse, you can try a compound called methyl phosphonic acid, very similar structurally to phosphoric acid and does well in organic solvents.
Mike
By HW Mueller on Thursday, February 13, 2003 - 12:02 am:
The convention is to state only the molarity of the aqu. part as the volume after mixing with organic is undefined (your case would be: 20 volume units phos. buffer 0.1M/pH4.4 + 80 vol. units ACN) Also, the final phosphate species and concentrations will always be the same for a given pH, no matter how you approach it (unless you add other buffering species). If your constellation causes precipitation you will have to go to another buffer if you want to have the same buffering capacity.
There have been lots of instructive discussions on buffers before.
By ananda on Thursday, February 13, 2003 - 06:32 am:
Thank you very much to both of you. It was very interesting that I found the answer from an old posting and at http://www.chromatography.co.uk/TECHNIQS/hplc/valves.htm
So bascially, what I found is phosphate precipitates in ACN specially when the ACN% is high and phosphate content is low. However, as even Dr. Muller and I also have seen, when the acetonitrile % is low, it doesnt precipitate (I checked his answer to the posting on Nov4, under "Buffer Problem". Sodium and phosphate behaves similary too.
Thanks again!
Ananda
By Anonymous on Thursday, February 13, 2003 - 05:05 pm:
//100 mL of 100mM KH2PO4 (pH 4.4) in 400 mL of ACN in order to prepare a 20 mM phosphate buffer in 80% acetonitrile//
There is no question of ppt. don,t add buffer to ACN
add Acetonitrile to buffer then you don;t have any promlum